Project Summary Next generation sequencing platforms have revolutionized modern approaches for understanding a wide variety of biological processes, including immune responses and cancer. However, the diversity of the cells involved in these processes has important implications for understanding biologic outcomes. For instance, the diversity of T cell receptors on lymphocytes during responses to virus or cancer can have dramatic effects on disease progression. Conversely, the diversity of cancer cells or virus populations has important implications for successful control of disease. Therefore, a critical hurdle in these situations is the ability to provide single-cell analysis techniques coupled with high-throughput next generation sequencing, to adequately measure the diversity of cells. Unfortunately, current single-cell analysis approaches are either unfeasible for large cell populations, too expensive, and/or require specialized equipment that is not available to most labs. To address this problem, we have engineered DNA origami nanostructures that are able to specifically bind and protect two different mRNA within transfected cells, and use novel molecular approaches facilitated by the constrained geometry of the mRNA bound to DNA origami to generate bi-cistronic amplicons for use in paired-end high-throughput next generation sequencing. Importantly, the mRNA from individual cells remain physically linked throughout this process, so linked sequences are from individual cells. In this proposal we develop this approach for quantitating the diversity of clonally-distributed TCR? and TCR? T cell receptors in lymphocyte populations. We have shown that we can transfect polyclonal populations of T cells with high efficiency, isolate DNA origami nanostructures with bound TCR mRNA from transfected T cells, and generate CDR3 amplicons for Illumina 2x250 paired-end deep sequencing reactions to obtain linked TCR? and TCR? sequence information from individual T cells without the need for single cell sorting. We propose to validate and use the developed DNA origami nanostructures to provide the first estimate of total TCR diversity in the nave T cell repertoires of mice. This technology will be useful for downstream application to a wide variety of biologic processes, by relatively simple modifications to the DNA origami nanostructure probe sequences, including single-cell analysis of other diverse lymphocyte populations, including other T cell subsets or antibody producing B cells, as well as single cells analysis of heterogeneous tumors or diverse microbial communities. Moreover, because this approach utilizes equipment found in most modern molecular biology laboratories, it can be easily adopted by many researchers for these analyses.